Directed evolution of microorganisms

ABSTRACT

The present invention provides methods for directing the evolution of microorganisms comprising the use of mutator genes and growth under conditions of selective pressure. The method discloses mutator genes which can be used in the methods of the present invention and provides ATCC deposits which exemplify the evolved microorganisms produced by the methods.

FIELD OF THE INVENTION

[0001] The present invention relates to methods for directing theevolution of microorganisms using mutator genes. Such methods provide apool of microbial genetic diversity advantageous for industrialapplication, such as for the industrial production of heterologousproteins, such as hormones, growth factors and enzymes, and thebiocatalytic production of chemicals, vitamins, amino acids and dyes.

BACKGROUND OF THE INVENTION

[0002] The industrial applicability of microorganisms is restricted bytheir physiological limits set by solvent, pH, various solutes, saltsand temperature. Organic solvents are generally toxic to microorganismseven at low concentrations. The toxicity of solvents significantlylimits use of microorganisms in industrial biotechnology for productionof specialty chemicals and for bioremediation applications. Solventmolecules incorporate into bacterial membranes and disrupt membranestructure (Isken and Bont, 1998, Extremophiles 2(3): 229-238); (Pinkartand White, 1997, J. Bacteriol. 179(13): 4219-4226); (Ramos, Duque etal., 1997, “J. Biol. Chem. 272(7): 3887-3890); (Alexandre, Rousseaux etal., 1994, FEMS Microbiol, Left, 124(1): 17-22); and Kieboom, Dennis etal., 1998, J. of Bacteriology 180(24): 6769-6772). Classic strainimprovement methods including UV and chemical mutagenesis have beenapplied for selection of more tolerant strains (Miller, J., “A ShortCourse In Bacterial Genetics,” Cold Spring Harbor Laboratory Press,1992). A number of studies have been dedicated to identification andisolation of solvent tolerant mutants among various bacterial strains.Spontaneous E. coli solvent tolerant mutants and mutants isolated in theprocess of 1-methyl-3-nitrosoguanidine (NTG) mutagenesis were obtainedfrom strain K-12 (Aono, Aibe et al., 1991 Agric. Biol. Chem 55(7):1935-1938). The mutants could grow in the presence of diphenylether,n-hexane, propylbenzene, cyclohexane, n-pentane, p-xylene. VariousPseudomonas strains were able to adapt and to grow in a toluene-watertwo-phase system (Inoue and Horikoshi, 1989, Nature 338: 264-266), withp-xylene (Cruden, Wolfram et al., 1992, Appl. Environ. Microbiol. 58(9):2723-2729), styrene and other organic solvents (Weber, Ooijkaas et al.,1993, Appl. Environ. Microbiol. 59(10): 3502-3504), (de Bont 1998,Trends in Biotechnology 16: 493-499). Yomano et al. isolated ethanoltolerant mutants which increased tolerance from 35 g/l to 50 μg/l during32 consequent transfers (Yomano, York et al., 1998, J. Ind. Microbiol.Biotechnol. 20(2): 132-138). High temperature evolution using E. colihas been disclosed in the art (Bennett, 1990, Nature, Vol. 346, 79-81)however the fitness gain was low as compared to the parent.

[0003] Strains of E. coli that carry mutations in one of the DNA repairpathways have been described which have a higher random mutation ratethan that of typical wild type strains (see, Miller supra, pp.193-211).As reported by Degenen and Cox (J. Bacteriol., 1974, Vol. 117, No. 2,pp. 477-487), an E. coli strain carrying a mutD5 allele demonstratesfrom 100 to 10,000 times the mutation rate of its wild type parent.Greener et al., “Strategies In Molecular Biology,” 1994, Vol. 7,pp.32-34, disclosed a mutator strain that produces on average onemutation per 2000 bp after growth for about 30 doublings.

[0004] Microorganisms are used industrially to produce desired proteins,such as hormones, growth factors and enzymes and to produce chemicals,such as glycerol and 1,3 propanediol (WO 98/21340 published May 22, 1998and U.S. Pat. No. 5,686,276 issued Nov. 11, 1997), vitamins, such asascorbic acid intermediates (1985, Science 230:144-149), amino acids,and dyes, such as indigo (U.S. Pat. No. 4,520,103, issued May 28, 1985).In spite of advances in the art, there remains a need to improve themicroorganisms and methods for producing such desired proteins,chemicals, amino acids and dyes.

SUMMARY OF THE INVENTION

[0005] The present invention relates generally to methods for directingthe evolution of a microoganism, that is for directing desired geneticchange in a microorganism in response to conditions of selectivepressure. In one aspect, the present invention relates to methods forevolving microorganisms to grow under extreme conditions, such as athigh temperature, under conditions of pH extremes, in the presence ofsolvents, and in the presence of high salt. In another aspect, thepresent invention relates to methods for evolving a microorganismcomprising at least one nucleic acid encoding a desired protein or anenzyme in an enzymatic pathway to grow under desired conditions.

[0006] The present invention is based, in part, upon the finding thatmicrorganisms such as wild-type E. coli and E. blattae, can be evolvedinto microorganisms capable of growing in the presence of high solvents,such as DMF and 1,3 propanediol, using methods described herein. Thepresent invention is also based, in part, upon the finding that E. colican be evolved into a microorganism capable of growing at elevatedtemperatures using methods described herein. The present invention isfurther based, in part, upon the identification of the optimal mutationrate for a microorganism and the discovery that the mutation rate can becontrolled.

[0007] Accordingly, the present invention provides a method forpreparing an evolved microorganism comprising the steps of culturing amicroorganism comprising at least one heterologous mutator gene for atleast 20 doublings under conditions suitable for selection of an evolvedmicroorganism, wherein said heterologous mutator gene generates amutation rate of at least about 5 fold to about 100,000 fold relative towild type, and restoring said evolved microorganism to a wild typemutation rate. In one embodiment, the microorganism further comprises atleast one introduced nucleic acid encoding a heterologous protein, saidprotein(s) including, but not limited to hormones, enzymes, growthfactors. In another embodiment, the enzyme includes, but is not limitedto hydrolases, such as protease, esterase, lipase, phenol oxidase,permease, amylase, pullulanase, cellulase, glucose isomerase, laccaseand protein disulfide isomerase. The present invention encompassesgenetic changes in the microorganism as well as changes in theintroduced nucleic acid.

[0008] In yet a further embodiment, the microorganism further comprisesintroduced nucleic acid encoding at least one enzyme necessary for anenzymatic pathway. In one embodiment, the introduced nucleic acid isheterologous to the microorganism; in another, the introduced nucleicacid is homologous to the microorganism. In a further embodiment, theenzyme is a reductase or a dehydrogenase and said enzymatic pathway isfor the production of ascorbic acid or ascorbic acid intermediates. Inan additional embodiment, the enzyme is glycerol dehydratase or1,3-propanediol dehydrogenase and said enzymatic pathway is for theproduction of 1,3 propanediol, 1,3 propanediol precursors or 1,3propanediol derivatives. In another embodiment, the enzyme isglycerol-3-phosphate dehydrogenase or glycerol-3-phosphate phosphataseand said pathway is for the production of glycerol and glycerolderivatives. In a further embodiment, the enzymatic pathway is for theproduction of amino acids, such as tryptophane or lysine or dyes, suchas indigo.

[0009] In one embodiment of the present invention, the microorganism iscultured for between about 20 to about 100 doublings; in anotherembodiment, the microorganism is cultured for between about 100 to about500 doublings; in yet another embodiment, the microorganism is culturedfor between about 500 to about 2000 doublings and in a furtherembodiment, the microorganism is cultured for greater than 2000doublings. In one embodiment, the mutator gene generates a mutation rateof at least about 5 fold to about 10,000 fold relative to wild type; inanother embodiment, the mutator gene generates a mutation rate of aleast about 5 fold to about 1000 fold and in another embodiment, themutator gene generates a mutation rate of about 5 fold to about 100 foldover wild type.

[0010] In one embodiment, an evolved microorganism comprises from about3 to about 1000 selected mutations in about 3 to about 500 genes and mayfurther comprises from about 20 to about 100,000 neutral mutations. Inone aspect, the mutations generated are non-specific and in anotheraspect, the mutations generated are specific.

[0011] In one embodiment of the present invention, the microorganismcomprises a plasmid comprising the heterologous mutator gene and saidstep of restoring said evolved microorganism to a wild type mutationrate comprises curing the evolved microorganism of said plasmid. Inanother embodiment, the plasmid comprises a temperature sensitive originof replication and the curing comprises growing the evolvedmicroorganism at a restrictive temperature. In a further embodiment, themicroorganism comprises at least one copy of the mutator gene in thechromosome and said step of restoring said evolved microorganism to awild type mutation rate comprises excision or removal of said mutatorgene from the host genome or the replacement of the mutator gene with afunctional (non-mutator) allele of the same gene.

[0012] In one embodiment, the present invention comprises the use of atleast one mutator gene to evolve a microorganism. In another embodiment,the mutator gene includes but is not limited to a mutD gene mutation, amutT gene mutation, a mutY gene mutation, a mutM gene mutation, a mutHgene mutation, a mutL gene mutation, a mutS gene mutation or a mutU genemutation and homologues of these DNA repair genes which have beenmutated as long as the mutated gene has impaired proofreading function.In a further embodiment, the mutator gene comprises at least one of themutD mutations disclosed herein in Table I.

[0013] In one embodiment of the present invention, conditions suitablefor selection include but are not limited to culturing saidmicroorganism in the presence of at least one organic solvent, such asfor example, alcohols, diols, hydrocarbon, mineral oil, mineral oilderived products, halogenated compounds and aromatic compounds; in thepresence of high temperature, such as in the range of 42°-48° C.; in thepresence of high salt, and in the presence of extreme pH conditions,such as alkaline or acidic conditions.

[0014] The present invention encompasses methods for evolving grampositive and gram negative microorganisms as well as yeast, fungus andeucaryotic cells including hybridomas. In one embodiment, the gramnegative microorganism includes members of Enterobacteriaceae and inanother embodiment comprises Eschericia and in another embodimentcomprises E. coli and E. blatte. In further embodiments of the presentinvention, the evolved microorganism includes E. coli having ATCCaccession number ______ and E. blatte having ATCC accession number______.

[0015] The present invention also provides expression vectors and hostcells comprising a mutator gene and methods for producing such vectorsand host cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 shows the nucleic acid and amino acid sequence of the mutDgene. Illustrative examples of mutations of the mutD gene are provided.

[0017]FIG. 2 provides the nucleic acid sequence for the enzyme1,3-propanediol dehydrogenase (PDD).

[0018]FIG. 3 provides the amino acid sequence for the enzyme1,3-propanediol dehydrogenase (PDD).

[0019]FIG. 4 provides a time course for E. coli cultures subjected todirected evolution and selection under elevated temperature.

[0020]FIG. 5—Glycerol fermentation of E. blattae at pH 7.0. Cultureconditions are described in the text. Plate counts were by serialdilution and performed in triplicate on Luria agar plates. Substrate andOproducts were measured by HPLC.

[0021]FIG. 6—Glycerol fermentation of E. blattae strain GEB031-4 at pH7.0. Culture conditions are described in the text. Plate counts were byserial dilution and performed in triplicate on Luria agar plates.Substrate and products were measured by HPLC.

DESCRIPTION OF THE MICROORGANISM DEPOSITS MADE UNDER THE BUDAPEST TREATY

[0022] Applicants have made the following biological deposits under theterms of the Budapest Treaty on the International Recognition of theDeposit of Micro-organisms for the Purposes of Patent Procedure:Depositor Identification International Reference Depository DesignationDate of Deposit Escherichia coli MM294 ATCC May 17, 1999 derivativeEscherichia blattae 33429 ATCC May 17, 1999 derivative

DETAILED DESCRIPTION Definitions

[0023] A mutation refers to any genetic change that occurs in thenucleic acid of a microorganism and may or may not reflect a phenotypicchange within the microorganism. A mutation may comprise a single basepair change, deletion or insertion; a mutation may comprise a change,deletion or insertion in a large number of base pairs; a mutation mayalso comprise a change in a large region of DNA, such as throughduplication or inversion.

[0024] When many possible different mutations in nucleic acid can giverise to a particular phenotype, the chance of mutation to that phenotypewill be higher than in a situation where only a few types of mutationscan give rise to a particular phenotype. As used herein the terms“wild-type mutation” and “spontaneous mutation” are usedinterchangeably. The rate of spontaneous mutation is defined as theprobability of a mutation each time the genome is replicated or doubled.As used herein “mutation rate” is simultaneous with “frequency” andrefers to the absolute number of mutations/doubling/base pair. As usedherein, the term “relative rate” refers to the ratio of mutation ratesof two strains, one of these is usually a wild type strain. Relativerate indicates how much more likely it is that a strain will undergomutation as compared to the wild type strain. The frequency ofspontaneous mutation of wild type E. coli (the E. coli genome has about4.6×10⁶ base pairs) is about 5×10⁻¹⁰ mutations per base pair perdoubling (see Drake, 1991). Doubling refers to the process ofreproduction of at least part of a genome of an organism and usuallyinvolves reproduction by binary fission. As used herein, “doubling”encompasses the reproduction of nucleic acid within an microorganismachieved by any means.

[0025] As used herein, a “mutator strain” refers to a microorganismhaving a higher than naturally occurring rate of spontaneous mutation.As used herein, “mutator gene” refers to a DNA repair gene whichcomprises a mutation and which has impaired proof reading function. Asused herein, the term “mutator plasmid” refers to a plasmid orexpression vector or cassette comprising a mutator gene. Culturing amicroorganism comprising a mutator gene will give rise to mutationalevents during genome replication. The present invention encompasses theuse of any DNA repair genes comprising mutations as long as the mutatedDNA repair gene is capable of introducing mutational events in amicroorganisms genome or on a gene introduced into the microorganism.DNA repair genes include but are not limited to, mutD, mutT, mutY, mutM,mutH, mutL, mutS or mutU and homologues of these genes. A homologue asused herein refers to a functionally related DNA repair gene. In oneembodiment, the mutator gene is a mutD gene (the epsilon subunit of DNApolymerase III, see Degnen et al., 1974, J. Bacteriol. 117:477-487)comprising mutations that provide an impaired proofreading function. Inone embodiment disclosed herein, the mutD mutation is introduced into amicroorganism on a plasmid. Illustrative embodiments of MutD mutationsare disclosed herein in Table I. The mutD mutations impair proofreadingfunction of the epsilon subunit of DNA polymerase III holoenzyme bysignificant decrease in the 3′-5′ exonuclease activity (Takano et al.,1986, Mol. Gen. Genet. 205(1):9-13).

[0026] When referring to mutations or genetic changes in an evolvedmicroorganism, “neutral mutation” refers to a mutation which has littleor no measurable effect on the phenotype of an evolved strain under agiven set of conditions. Examples of “neutral mutations” include, butare not limited to, silent mutations which do not lead to a change inthe amino acid sequence of the encoded protein, mutations which affectproteins that are not essential for growth under a given set of cultureconditions, and mutations in non-coding regions of the chromosome. Inone illustrative embodiment herein, an E. coli strain evolved for hightemperature was characterized as being auxotrophic for three amino acids(ie, were not able to grow in medium without Cys/Met, Asp/Asn, and Pro)indicating that there were at least three neutral mutations in the E.coli in addition to the mutations associated with growth at hightemperature. The term “selected mutation” as used herein refers to thosemutations which are associated with a phenotype of an evolved strainunder a given set of conditions. Being associated with means that themutation is directly or indirectly responsible for the improved oraltered phenotype.

[0027] When referring to mutations or genetic changes in a host cell ormicroorganism, nonspecific refers to the changes in the host cell genomewhich occur randomly throughout the genome and which potentially canaffect all bases and includes frameshifts. Non-specific mutationsencompass changes in single base pairs as well as changes in a largenumber of base pairs as well as changes in large regions of DNA. Forexample, in one embodiment, an evolved microorganism which has beenexposed to a MutD gene comprising mutations that impair the proofreading function will comprise random mutations at a rate of about5-1000 times over wild type. In one illustrative embodiment of themethod using a mutD mutation, the evolved strain had at least 3 randommutations. The present invention encompasses any rate of mutations thatprovides the desired phenotype. When referring to genetic changes in ahost cell, specific mutation refers to mutations which can becharacterized or which comprise definable genetic changes, such as A:Tto C:G transversion characteristic of mutT mutations; G:C to T:Atransversion characteristic of mutY and mutM mutations; A:T to G:C andG:C to A:T transitions and frameshifts characteristic of mutH, mutL,mutS, uvrD (mutU) mutations; G:C to T:A transversions characteristic ofthe mutYmutM double mutation (Miller et al., A Short Course in BacterialGenetics, a Laboratory Manual and Handbook for E. coli and RelatedBacteria).

[0028] When referring to a mutator gene, “heterologous” means that thegene is introduced into the cell via recombinant methods and ispreferably introduced on a plasmid. The mutator gene may also beintroduced into the microorganism genome through recombinant techniques.The mutator gene introduced into the microorganism may be a mutation ofa naturally occurring DNA repair gene in the cell or may be foreign tothe host microorganism. Referring to nucleic acid as being “introduced”into a microorganism means that the nucleic acid is put into themicroorganism using standard molecular biology techniques. An introducednucleic acid may be the same or different than nucleic acid naturallyoccurring in the microorganism.

[0029] As used herein the term “restoring to wild type mutation rate”refers to the process whereby a mutator gene is removed from an evolvedmicroorganism thereby restoring the wild-type mutation rates. Thepresent invention encompasses any process for removing the mutator genefrom an evolved organism and includes but is not limited to curing theorganism of a resident plasmid comprising the mutator gene or byexcising or otherwise removing the mutator gene from the host genomesuch that normal DNA repair function is restored. Curing refers tomethods for producing cells which are free of a plasmid comprising themutator gene. A microorganism can be cured of any resident plasmid usingtechniques known to the skilled artisan.

DETAILED DESCRIPTION

[0030] One of the basic tenants of inheritance is that mutations occurrandomly and then are selected by the environment. Mutations that happento confer a selective advantage on the organism are preferentiallypassed on to future generations. The present invention relates tomethods for directing desired genetic change in a microorganism, iedirecting the evolution of a microorganism, by exposing themicroorganism to a mutator gene, selecting for acquisition of desiredcharacteristics in the evolved microorganism, and curing themicroorganism of the mutator gene, or otherwise removing the mutatorgene, such that wild type mutation rates are restored.

I. Uses of the Invention

[0031] In one aspect of the present invention, the methods are used toevolve a microorganism to grow under extreme conditions, such as in thepresence of elevated temperature, high solvent, altered pH or in thepresence of high salt. In another aspect of the present invention, themethods are used to evolve microorganisms which comprise introducednucleic acid encoding a heterologous protein or at least one enzyme inan enzymatic, ie biocatalytic pathway. Such commercially importantproteins include hormones, growth factors and enzymes. Illustrativebiocatalytic pathways include those disclosed in U.S. Pat. No.5,686,276, issued Nov. 11, 1997, for the production of 1,3-propanedioland in 1985, Science 230:144-149 for the production of ascorbic acidintermediates.

[0032] Methods of the present invention are especially advantageous forproducing improved microorganisms used for the biocatalytic productionof chemicals and vitamins where numerous catalytic events are takingplace either concurrently or sequentially within the host microorganism.In such complex biocatalytic systems, it is often difficult to identifythe specific molecular events causing low yields, host toxicity orcatalytic failures and therefore difficult if not impossible tounderstand which specific genetic events to alter in order to correctthe deficiencies. The methods of the present invention provide theadvantage of allowing the microorganism to make the required changes inresponse to selective pressure.

[0033] Additionally, the methods of the present invention provide anadvantage for obtaining microorganisms comprising desired phenotypictraits associated with multiple genes, such as the ability of amicroorganism to grow at elevated temperatures. The use of the mutatorgene provides a means for producing genetic diversity and thesimultaneous growth under conditions of selective pressure allows themicroorganism to identify the specific genetic changes required forsurvival. The use of mutD gene mutations allows for very large diversityto be provided to the microorganism from which to select for thespecific genetic changes that provide a growth advantage. Therefore, themethods disclosed herein avoid the limited diversity that is oftenproduced with art methods that begin the directed evolution process withdefined sets of genes. Furthermore, the methods disclosed hereineliminate additional screening steps often associated with art methodsfor producing genetic diversity. A further advantage of the presentinvention is that the methods can be applied to microorganisms whichhave not been sequenced and for which there may be limited informationupon which to design genetic changes.

[0034] In illustrative embodiments disclosed herein, a mutated mutD generesiding on a plasmid was introduced via recombinant techniques into E.coli or E. blatte. The E. coli or E. blatte cell was then cultured underconditions suitable for growth for a time sufficient for at least 20doublings and up to at least about 2000 doublings under conditions ofselective pressure. In one example, E. coli was grown under conditionsof increased temperature or in the presence of DMF and in another E.blattae was growth in the presence of solvent, such as DMF or 1,3propanediol. As a result, E. coli was evolved into a microorganismcapable of growing at temperatures up to about 48° C. or in the presenceof 80 g/l DMF. E. coli evolved to grow at elevated temperatures alsobecame auxotrophic for three amino acids, Cys/Met, Asp/Asn and Pro. E.blattae was evolved into a microorganism capable of growinganaerobically in the presence of at least 105 g/l 1,3-propanediol andwhich comprised genetic changes in at least one catalytic activityassociated with 1,3 propanediol production, 1,3-propanedioldehydrogenase, shown in FIG. 3.

[0035] The use of a plasmid comprising a mutator gene, ie, a mutatorplasmid, can be used to control the mutation rate of a microorganism. Asdescribed under Section II below, plasmid constructs can be designedwhich provide reduced levels of expression of a mutator gene therebyproviding a means for altering the ratio of naturally occurring DNArepair genes vs mutator genes. This provides a means for combining theadvantage of mutD mutations (which results in random mutagenesis) withthe advantages of the other known mutators (lower mutation frequencywhich leads to a lower burden on the cells). Additionally, plasmidconstructs can be designed that allow for curing the evolvedmicroorganism of the mutator gene, such as the use of a temperaturesensitive origin, thereby allowing for a means for turning the mutationevents off and on in the microorganism. For a gram positivemicroorganism, such as B. subtilis where the entire genome has beensequenced, the present invention could encompass the steps of deletingor mutating a DNA repair gene, evolving the Bacillus, and restoring thenaturally occurring DNA repair system through recombination events. Asdisclosed herein, several members of Escherichia, such as E. coli and E.blatte have been subjected to the directed evolution methods.Illustrative examples of evolved E. coli and E. blattae have beendeposited with the ATCC and have accession numbers, ______ and ______,respectively.

[0036] The methods of the present invention provide a means toaccomplish long-term evolution of microorganisms. An E. coli straincomprising a plasmid comprising a mutD mutation was grown for >1000doublings without a reduction in mutation rate. The present inventionalso provides a means for reducing the functional genome of an organism.A microorganism can be grown for many thousands of generations, suchthat only the genes which are essential would remain functional. Most ofthe other genes would carry random and inactivating mutations.

[0037] The present invention also provides a means for makingnon-pathogenic organisms. A pathogenic strain can be evolved into amutator strain by introduction of a mutator gene and grown for extendedperiods of time. As a result many of the genes that are involved inpathogenicity would become inactivated and the strain would be safe touse.

[0038] The present invention also provides a means to streamline themetabolism of an organism. A strain which has an improved yield onnutrients or a reduced metabolic rate (maintenance metabolism) can beproduced by methods disclosed herein. Such strains would be usefulproduction strains for chemicals as well as enzymes. The presentinvention provides a means for making microorganisms mutator strains byintroducing a mutator gene, thereby protecting the microorganism'snaturally occurring DNA repair genes from becoming mutator genes inresponse to selective pressure. That is, the introduction of the mutatorplasmid into a microorganism whether via a plasmid or into the genome,protects the cells from developing a mutator phenotype in response toselective pressure.

II. Mutator Genes and Frequency of Mutations

[0039] Mutator genes of the present invention include but are notlimited to, mutations of the DNA repair genes mutD, mutT, mutY, mutM,mutH, mutL, mutS or mutU or their homologues in other microorganisms. Adescription of the DNA repair genes are disclosed in Miller, supra; mutDis disclosed in Maki et al., 1983, Proc. Natl. Acad. Sci., U.S.A. 80,7137-7141 (GenBank accession number K00985.1 GI: 147678 and FIG. 1); B.subtilis mutS and mutL are disclosed in Ginetti et al., 1996,Microbiology, August, 142 (Pt 8): 2021-9; Streptococcus pneumoniae hex Brepair gene, mutL of Salmonella typhimurium and PMS1 of Saccharomycescerevisiae are disclosed in Prudhomme et al., 1989, J. Bacteriology,October; 171 (10): 5332-8; Streptococcus pneumoniae hexA and mutS ofSalmonella typhimurium and E. coli are disclosed in Priebe et al., J.Bacteriol, 1988, January; 170(1): 190-6 and Prudhomme et al., 1991, J.Bacteriol. November; 173(22): 7196-203; human mutS homologue, hMSH2, andhuman MutL homologue, hMLH1, are disclosed in Macdonald et al., 1998,Heptology, July 28(1):90-7; the mut-1 of Neurospora is disclosed inDillon et al.,1994, Genetics, September 138(1):61-74 and yeasthomologues of mutL and mutS are disclosed in WO 97/15657. The methods ofthe present invention comprises the use of at least one of the mutantDNA repair genes and may involve the use of more than one. It ispreferred that a mutator gene be dominant to the wild type gene of themicroorganism such that mutations are introduced into the genome of themicroorganism. In a preferred embodiment, the mutator gene is a mutationof the mutD gene. The nucleic acid and amino acid sequence for mutD isshown in FIG. 1. One particular mutD mutation, mutD5, is disclosed inTakano, K., et al., (1986, Mol Gen Genet 205, 9-13, Structure andfunction of dnaQ and mutD mutators of Escherichia coli). Strain CSH116was obtained as disclosed in Miller, J. H. (1992, A Short Course inBacterial Genetics). This strain is reported to carry the mutD5 allele.The mutD gene in this strain was found to be very different from thepublished mutD5. The mutD gene from strain CSH116 is designated hereinas mutD5′. Table I gives the mutations found in mutD5 and mutD5′.Further mutations in mutD which result in increased levels of mutationfrequency were identified recently in Taft-Benz, S. A. et al., (1998,Nucl. Acids Res. 26, 4005-4011, Mutational analysis of the 3′-5′proofreading exonuclease of Escherichia coli DNA polymerase III). TableI describes various mutD mutations useful in the present invention.Table II describes various promoters used with the mutD mutations andTable III describes mutator plasmids and the range of available mutationfrequencies in E. coli. TABLE I mutations in the coding region of mutDMutD Clone #nucleo- #amino amino amino tide acid nucleotide acidnucleotide acid  44  15 C Thr T Ile mutD5′ 218  73 T Leu G Trp mutD5 369123 T Thr C Thr mutD5′ 418 138 C Pro T Pro mutD5′ 451 151 T Ala C AlamutD5′ 484 161 G Leu A Arg mutD5′ 491 164 C Ala T Val mutD5 661 220 AGlu C Asp mutD5′ 665 222 A Ile C Leu mutD5′ 673 225 T Ala A Ala mutD5′688 228 C Leu T Leu mutD5′ 706 236 A Lys G Lys mutD5′ 715 239 T Ser CSer mutD5′ 727 243 A Arg G Arg mutD5′

[0040] TABLE II mutD Mutations Name Mutations wild type ATGACCGCTATG...pOS100 TTG A-CGCT TTG... pOS101 GTGACCGCT GTG... pOS102 GTG-CCGCT GTG...pOS104 TTGACCGCT TTG... pOS105 GTGACCGCTGTGAGCACTT(G)CAATTAcACGCCAGATCGTTCTCGATACCGAAAT(C)... pOS106GTGACCGCT-TG...

[0041] TABLE III Mutator (mutD5) and control (mutD) plasmids and therange of available mutation frequencies in E. Coli. mut. Frequencymutator rate # plasmid genotype ori ab resistance size (kb) (average)(relative)  1 pMutD5-61 mutD5′ pSc kan 5.97  6.4 × 10⁻⁵ ˜1000-fold  2pMutD71-Ts mutD pSc kan 5.97  2.5 × 10⁻⁸ wild type  3 pBRmutD68 mutD5′pBR322 kan, bla 6.16  1.1 × 10⁻⁴ (Al data) ˜10000-fold  4 pBRmutD727mutD pBR322 kan, bla 6.16 nd wild type Modified  5 pOS100 mutD5′ pBR322kan, bla 6.16   2 × 10⁻⁵ ˜800-fold  6 pOS101 mutD5′ pBR322 kan, bla 6.16 3.8 × 10⁻⁶ ˜152-fold  7 pOS102 mutD5′ pBR322 kan, bla 6.16  1.1 × 10⁻⁶˜44-fold  8 pOS104 mutD5′ pSc kan 5.97 4.35 × 10⁻⁷ ˜17-fold  9 pOS105mutD5′ pSc kan 5.97  1.1 × 10⁻⁶ ˜44-fold 10 pOS106 mutD5′ pSc kan 5.97  5 × 10⁻⁶ ˜200-fold

[0042] MutD mutations can introduce all types of base pair changesincluding frame shifts (Miller, supra). MutD5 has a reported relativemutation frequency of 1000-10000 fold in rich medium, ie, 5×10⁻⁶ to5×10⁻⁷ mutations per doubling per base pair (Denegen et al., 1974, J.Bacteriol. 117, 477-478). Considering that the E. coli genome has4.6×10⁶ bp, then a mutD5 gene will generate 2.3 to 23 mutations perdoubling per genome. In a preferred embodiment of the present invention,mutator plasmids have been generated which allow for reduced expressionlevels of the mutated mutD repair gene such that the mutation raterelative to wild type is reduced. As illustrated in Table II, the MutDgene has 2 closely located ATG start codons with 6 nucleotides betweenthem. The first ATG is considered to be putative. Both ATG codons werereplaced with GTG or TTG codons for reducing mutD5′ expression levels.The space between the 2 ATG codons up to 5 nucleotides was truncated.The plasmids comprising these mutator genes provide lower mutation rateswhen introduced in E. coli in comparison to MutD5′ plasmids.

[0043] As a result, the microorganism comprising the plasmid comprisingthe mutated mutD gene expressed normal levels of the functional epsilonsubunit coded by naturally occurring mutD and low amounts of thenon-functional epsilon subunit coded by the mutated mutD5′. Bothsubunits compete for polymerase III. Consequently, the microorganismwill most of the time have functional proof-reading but to a certainfraction of time the cell will copy its DNA without proof-reading, dueto the presence of the mutD mutations. Thus, the frequency ofmutagenesis of a microorganism can be altered by adjusting theexpression of the mutator gene or by altering the ratio of a naturallyoccurring DNA repair gene to the corresponding mutated DNA repair gene.The mutations caused by these plasmid introduced mutator genes shouldstill be as random as mutations caused by a chromosomal copy of mutD5.Data generated in Example III indicate that mutation rates of 5-1000times over wild type are preferred for most applications. Other means ofcontrolling the mutation frequency include having two copies of mutD onthe plasmid or integrated into the microrganism genome and/or using atransmissible heat sensitive plasmid which could be used to temporarilytransform cells into mutators and then restore them to wild type rates.Yet another way to adjust the mutation frequency is to identify mutDmutations which result in moderate mutation frequency due to reducedproof reading. Such mutants have been recently identified but it is notknown if these mutations preferentially result in a few types ofspecific mutations, Taft-Benz et al., 1998, Nucl. Acids. Res.26:4005-4011. Mutation rates are determined using rifampicin orstreptomycin as disclosed in Horiuchi, et al., 1978, Mol. Gen. Genet.163:227-283.

[0044] Mutation rates and a description of the molecular fingerprint ofa microorganism produced by the methods disclosed herein and claimed arealso exemplified by virtue of the microorganism deposits made with theATCC under the terms of the Budapest treaty.

III. Construction of Mutator Genes and Mutator Plasmids

[0045] Construction of plasmids comprising mutator genes andtransformations of microorganisms can be performed by means deemed to beroutine to the skilled artisan. In one embodiment illustrated herein,nucleic acid encoding a mutator gene is introduced into a microorganismon a replicating plasmid, ie, a mutator plasmid, which is cured orotherwise eliminated from the microorganism after evolution. In anotherembodiment disclosed herein, nucleic acid encoding a mutator gene isintroduced into a microorganism's genome in addition to or as areplacement of a naturally occurring DNA repair gene.

[0046] Nucleic acid encoding a mutator gene can be isolated from anaturally occurring source or chemically synthesized as can nucleic acidencoding a protein or enzyme. Sources for obtaining nucleic acidencoding DNA repair genes mutD, mutT, mutY, mutM, mutH, mutL, mutS ormutU is provided in Section II. FIG. 1 provides the nucleic acid andamino acid sequence for mutD and Table I and III provide preferredmutations for mutD and the mutation rates obtained for each construct.Once nucleic acid encoding a mutator gene is obtained, plasmids or otherexpression vectors comprising the mutator gene may be constructed usingtechniques well known in the art. Molecular biology techniques aredisclosed in Sambrook et al., Molecular Biology Cloning: A LaboratoryManual, Second Edition (1989) Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989) and Brown, T. Current Protocols in MolecularBiology, Supplements 21, 24, 26 and 29. Nucleic acid encoding a mutatorgene is obtained and transformed into a host cell using appropriatevectors. A variety of vectors and transformation and expressioncassettes suitable for the cloning, transformation and expression inbacteria are known by those of skill in the art.

[0047] Typically, the plasmid vector contains sequences directingtranscription and translation of the nucleic acid, a selectable marker,and sequences allowing autonomous replication or chromosomalintegration. Suitable vectors comprise a region 5′ of the gene whichharbors transcriptional initiation controls and a region 3′ of the DNAfragment which controls transcriptional termination. These controlregions may be derived from genes homologous or heterologous to the hostas long as the control region selected is able to function in the hostcell.

[0048] Initiation control regions or promoters, which are useful todrive expression of the mutator gene. Virtually any promoter capable ofdriving expression is suitable for the present invention. Once suitablecassettes are constructed they are used to transform the host cell.General transformation procedures are taught in Current Protocols InMolecular Biology (Vol. 1, edited by Ausubel et al., John Wiley & Sons,Inc., 1987, Chapter 9) and include calcium phosphate methods,transformation using PEG, electroporation and protoplast transformation.

[0049] After subjecting a microorganism to directed evolution using amutator plasmid, the microorganism is cured of the mutator plasmid inorder to restore the microorganism to wild-type mutation rates. Methodsfor curing a microorganism of a resident plasmid comprising a mutatorgene include transformation of the microorganism comprising a mutatorplasmid with an incompatible plasmid; electroporation techniques asdescribed in Heery et al., 1989, Nucl. Acids. Res., 17: 10131; growthwith acridine orange or ethidium bromide in the medium (Jeffrey Miller,1972, in Curing of Episomes from E. coli strains with Acridine Orangefrom Experiments in Molecular Genetics, Cold Spring Harbor Laboratories,pg. 140). In this method, acridine orange is added to 5 ml cultures ofan Enterobacteriaceae strain at 125 μg/ml and allowed to grow overnightat 37° C. The following day, the cultures are plated out and individualcolonies are used to prepare plasmid nucleic acid. The nucleic acid isanalysed by means known to those of skill in the art to determine thepresence or absence of the plasmid. For microorganisms comprising amutator gene in their genome, techniques known to those of skill in theart can be used to restore the microorganism back to wild-type mutationrates, such as excising the mutator gene or replacing the mutator genewith the naturally occurring DNA repair gene through homologousrecombination techniques.

IV. Culture Conditions and Selective Pressure

[0050] Once a microorganism has been exposed to a mutator gene, it iscultured under conditions of desired selective pressure, such aselevated temperature, pH, salt or in the presence of a solvent, such as,for example, DMF or 1,3 propanediol. Examples of other solvents includealcohols, diols, hydrocarbon, mineral oil, mineral oil derived products,halogenated compounds and aromatic compounds.

[0051] As the skilled artisan will appreciate, growth conditions arestrain dependent. General growth conditions are disclosed in Truesdellet al., (1991, Journal of Bacteriology, 173: 6651-6656) and Sonoyama etal. (1982, Applied and Environmental Microbiology, Vol. 43,p.1064-1069). Culture media may be supplemented when selectable markersare present such as antibiotic resistance genes, including but notlimited to tetracycline, ampicillin or chloramphenicol.

[0052] For the methods of the present invention, cultures may be grownaerobically or anaerobically in either liquid medium or solid medium,depending upon the microorganism and the type of selection. If culturesare grown in liquid medium, it is preferred to undergo a number ofrounds of replication (20 or more) in order to allow the survivors ofthe selection to grow over the wild-type. If cultures are grown in solidmedium, such as on an agar plate, it is preferred to have a number ofrepetitive platings in order to pick the survivors directly and to applyhigher selection pressure in each round and to amplify the populationthat is able to grow under the specific conditions of selection.

[0053] The manner and method of carrying out the present invention maybe more fully understood by those of skill in the art by reference tothe following examples, which examples are not intended in any manner tolimit the scope of the present invention or of the claims directedthereto.

EXAMPLES Example 1 Construction of mutD and mutD5′ Plasmids and Testingin 3 Bacterial Strains

[0054] The following example illustrates the construction of plasmidscomprising the mutator gene, mutD5′.

[0055] mutD and mutD5′ genes were amplified by PCR using mutd1(5′-CGCCTCCAGCGCGACAATAGCGGCCATC-3′) and mutd2(5′-CCGACTGAACTACCGCTCCGCGTTGTG-3′) primers from genomic DNA of E. coliand E. coli CSH 116 (Miller 1992), respectively. The PCR products werecloned into pCR-Blunt vector (Invitrogen, Carlsbad, Calif.). Plasmidsfrom clones with the correct orientation were isolated and digested withSmal-HindIII restriction enzymes. The overhang ends were filled using T4polymerase and cloned into pMAK705 plasmid digested with Smal-PvuII. Theligation products were transformed into JM101 competent cells. Theresulted plasmids had the temperature-sensitive origin of replication,carried kanamycin resistance marker and were named pMutD-71 (controlplasmid, wild type genotype) and pMutD5-61 (mutator plasmid).

[0056] The plasmids were successfully tested in E. coli MM294 (F⁻ endA1hsdR17 (r_(k) ⁻ m_(k) ⁺)supE44 thi-1 relA1) and E. blattae ATCCaccession number 33429 for evolution of solvent tolerance. All evolutionexperiments were performed in LB medium. Mutation rates were determinedby plating aliquots of cell suspensions on LB plates containing 100ug/ml rifampicin or streptomycin. The mutation frequency was calculatedby dividing the number of resistant cells by the total number of platedcells.

Example 2 Evolution of Solvent Tolerance

[0057] The following example illustrates the evolution of solventtolerant microorganisms using the mutator plasmids constructed as inExample 1.

[0058] In order to make evolution experiments quantitative, LB agarplates supplemented with 50, 60, 70, 80 and 90 g/l DMF and 25 ug/mlkanamycin were used. The size of every evolving population was limitedto 10⁶ cells. After each plating, the number of raised colonies wascounted and 10 were selected for the next plating. Cells from selectedcolonies were mixed together and aliquots containing 10⁶ cells wereplated on fresh plates containing the same and higher concentrations ofDMF. After 2 consequent platings the cells were cured of the plasmids bygrowth at elevated temperatures. E. blattae 33429 and E. coli MM294 werecured at 41° C. and 43° C., respectively. Three to four subculturings atindicated temperatures were sufficient for 87-100% curing. Individualcured clones were selected by parallel growth of clones in selective andnon-selective medium. The curing was also confirmed by plasmidpurification from selected clones and gel analysis.

[0059] The cured strains were tested for growth with the same DMFconcentrations as their plasmid containing parents. The experimentdemonstrates (1) the advantage of strains harboring mutator plasmidsover strains carrying control plasmids and (2) the preservation ofevolved features in strains cured from mutator plasmids.

[0060] The results of the short-term evolution are summarized in TableIV. In the process of 2 platings we obtained E. coli colonies, whichwere able to grow on plates containing 20 g/l higher concentration ofDMF than control clones. Analysis of E. coli MM294 harboring control andmutator plasmids revealed that the mutation frequency of cells carryingcontrol plasmids was more then 3 orders of magnitude lower in comparisonwith cells containing mutator pMutD5-61. Our results showed thathypermutation was very beneficial for cell survival at elevated DMFconcentrations (Table V). E. blattae 33429 appeared to be more sensitiveto DMF. Population of 10⁶ cells raised 968 colonies during 2 plating.When 10 bigger colonies were mixed and a new aliquots of 10⁶ cells wereplated on DMF plates supplemented with 70 g/l DMF, more than 1000 tinycolonies grew on the plates. However, these small colonies were notviable after transfer on fresh 70 g/l DMF plates. The mutation frequencyof E. blattae 33429 (pMutD5-61) dropped from 4.55×10⁻⁶ to 1.1×10⁻⁷ aftersecond plating on plates containing 60 g/l DMF (Table V). PlasmidMutD5-61 initially provided lower mutation frequency in E. blattae incomparison with E. coli. The E. blatte strain distinctly reducedmutability after cultivation in the presence of DMF. Although E. coliand E. blattae strains belong to the family Enterobacteriaceae, thebehavior of E. coli mutD5 gene product could be somewhat different in E.blattae cell environment. Nevertheless, the benefits of pMutD5 forsurvival of E. blattae on 60 g/l DMF plates were obvious. Control cellscarrying pMutD-71 couldn't grow in the presence of DMF above 50 g/l.

[0061] Contrary to E. blattae 33429 (pMutD5-61), we did not observedsignificant adjustments of mutability in E. coli strains. The mutationfrequency stayed within the same range at the end of evolutionexperiment. (Table V).

[0062] Single colonies of evolved cultures were used for curingexperiments. The curing efficiency was 87-100% with E. blattae 33429 andE. coli MM294. The mutation frequencies of cured clones were similar towild type control frequencies, and cured clones preserved their abilityto grow at elevated DMF concentration. E. blattae 33429 cured clonesgrew with 60 g/l DMF and E. coli MM294 grew with 80 g/l DMF. Initialtolerance of MM294 and E. blattae 33429 was 60 g/l and 50 g/l DMFrespectively. The evolved strains increased their tolerance by 20 g/land 10 g/l DMF, respectively. Therefore, sensitivity to DMF is straindependent.

[0063] The advantage of mutator plasmids for evolution in liquid culturewas tested as well. Within 4 days of solvent tolerance evolution inliquid medium supplemented with DMF or ethanol, E. blattae 33429(pMutD5-61) demonstrated growth at higher concentrations of bothsolvents in comparison with control cultures.

[0064] Mutator plasmids can be applied for evolution of bacterialtolerance to different solvents, various environmental stress andpotentially toxic specialty chemicals of industrial biotechnology. Oneadvantage of the directed evolution methods disclosed herein is that theevolution of microorganisms carrying mutator plasmids can be stopped atany time. Mutator plasmids can be cured from evolving strains, andtherefore, evolved desired features of the whole strain can bepreserved. TABLE IV Evolution of solvent tolerance. Colony formation byresistant clones on LB plates supplemented with various DMFconcentrations. Plating 1 Plating 2 DMF Number Number Strain Genotype(g/l) of colonies* of colonies* MM294(pMutD5-61) Mutator 60 g/l lowdensity high density lawn lawn MM294(pMutD5-61) Mutator 70 g/l  11 824MM294(pMutD5-61) Mutator 80 g/l  0  4 MM294(pMutD-71) Wild type 60 g/l 17 low density lawn MM294(pMutD-71) Wild type 70 g/l  0  0EB33429(pMutD5-61) Mutator 50 g/l low density high density lawn lawnEB33429(pMutD5-61) Mutator 60 g/l  0 968 EB33429(pMutD-71) Wild type 50g/l 793 high density lawn EB33429(pMutD-71) Wild type 60 g/l  0  0

[0065] TABLE V Mutation frequencies of bacteria harboring mutator andcontrol plasmids. Mutation rate Mutation rate Strain before theevolution DMF (g/l)* after the evolution MM294  9.2 ± 6.5 × 10⁻⁵ 80 g/l  4.7 ± 4 × 10⁻⁵ (pMutD5-61) MM294 4.15 ± 3.4 × 10⁻⁸ 60 g/l 2.9 ± 2.4 ×10⁻⁸ (pMutD-71) EB33429 4.55 ± 3.7 × 10⁻⁶ 60 g/l 1.13 ± 0.9 × 10⁻⁷ (pMutD5-61) EB33429  2.6 ± 2.1 × 10⁻⁸ 50 g/l 4.7 ± 3.8 × 10⁻⁸ (pMutD-71)

Example 3 Evolution of High Temperature Strains

[0066] Example 3 illustrates high temperature evolution under conditionsof continuous fermentation in the mode of turbidostat which allows forfermentation wherein the cell density is stabilized. Two independentexperiments were run with the strains: A: W1485 (ATCC12435) (=nonmutator); B: W1485/pBRmutD68 (same strain but comprises mutatorplasmid). Both strains were gown in continuous culture in a turbidostatin LB medium for about 1800 doublings. The temperature was controlled bya computer based on the measured growth rate to maintain a doubling timeof about 1 h. Whenever the culture grew faster the temperature wasraised and vice versa. The time course of both cultures is shown in FIG.4. Initially, the culture started from W1485/pBRmutD68 evolved fasterthan the culture started from W1485. This indicates the advantage of themutator plasmid. However, After about 400 doublings W1485 reached thehigher temperature. We also measured the mutation rate of samples takenfrom both evolution experiments. Table 6 shows that the starting clonesdiffered in their mutation frequencies by a factor of 3000. However,during the evolution experiments the mutation frequencies converged towithin a factor of 2. The experiment illustrates that the rate oftemperature evolution slows down over the course of the experiment. Itcan be expected that in a wild type strain there are a small number ofgenes which initially limit growth at elevated temperature. Favorablemutations in these genes will lead to relatively large gains in fitness.However, with increasing temperature more and more genes can be expectedto limit growth and the pace of evolution slows down. If individualmutations result in only very small growth benefits to their carrierthen the populations have to be grown for a significant number ofdoublings to enrich the clones carrying these mutations from thepopulation. As a consequence the optimum mutation rate for evolutionwill decrease during the process of evolution. For Table VI, mutationrates were determined using rifampicin as given in Miller (1992, supra).TABLE VI Mutation rates of strains and populations used for temperatureevolution Strain/population doublings temperature ° C. mutation rateW1485/pBRmutD68 0 45 3142.9 AL018 210 47.22 3428.6 AL019 376 47.502028.6 AL038 1811 48.21 1142.9 W1485 0 45 1.0 AL017 231 47.10 0.9 AL035543 47.91 134.3 AL040 1385 48.57 514.3

Example 4 Directed Evolution of E. blattae and Selection in the Presenceof a Solvent, 1,3-propanediol

[0067] E. Blattae ATCC accession number 33429 was transformed withplasmid pMutD68 (see Table III) and cultured in media containing 1, 5,10, 20, and 30 g/l 1,3 propanediol (cultures are designated as GEBxxxwhere “xxx” indicates the number of transfers into fresh media). Alldirected evolution experiments were carried out under anaerobicconditions in defined minimal medium with glycerol as a sole carbonsource. E. blattae doesn't require vitamin B₁₂ for growth, nevertheless,initial experiments were performed in 2 conditions (1) with B₁₂, and (2)without B₁₂ in the growth medium.

[0068] Within 18-22h GEB001 reached maximum 1030-1060 mOD (A₆₅₀) at allconcentrations of 1,3 propanediol. Therefore, 30 g/l 1,3-PD was notinhibitory for GEB001 growth. Growth rates of GEB in the presence of 50g/l were ˜½ of growth rates in the presence of 30 g/l 1,3-PD (590 mOD:1030 mOD in 22h). The threshold of tolerance to 1,3-PD was found between70 to 80 g/l. After 10 transfers, GEB010 was able to grow in thepresence of 80 g/l 1,3-PD to 350 mOD within 78h. However, these cellsfailed to grow at 80 g/l 1,3-PD concentration after next transfer.

[0069]E. blatte is known in the art to carry the enzymatic pathway forthe production of 1,3 propanediol (Roth, et al., 1986, Annu. Rev.Microbiol. 50:137-181). In order to determine if E. blatte can make1,3-propanediol in addition to the concentrations of 1,3 propanedioladded to the medium, GEB011 was grown in medium supplemented with 2-¹³Cglycerol and 70 g/l 1,3-PD. The supernatant was then analyzed by NMR(¹³C) and the results indicated the formation of ˜2.6 g/l ¹³C 1,3-PD.Therefore, GEB cells can make 1,3 propanediol in the presence of 1,3-PD.

[0070] The evolution of 1,3-propanediol resistance was faster in thepresence of B12. After 2 months of evolution GEB025 (+B12) was able togrow with 95-100 g/l 1,3-propanediol. After 3 months of anaerobic growthunder selection in the presence of 1,3-propanediol, GEB028 (-B12) couldgrow in medium supplemented with 110 g/l 1,3-propanediol. Analysis ofaerobic growth of GEB031 on LB plates supplemented lo with 85, 95, 105and 115 g/l 1.3-propanediol showed that cells produce much biggercolonies in the presence of 85 g/l in comparison with 105 g/l. No growthwas observed at 115 g/l 1,3 propanediol. The results indicate that after3 months of applying directed evolutions techniques described herein toE. blatte, the tolerance to 1,3 propanediol was increased from 75 g/l toat least 105 g/l under aerobic conditions. The plasmid was cured fromthe GEB031 strain by growing at 41.5 degrees. An illustrative clone,GEB031-4 was deposited with the ATCC and has accession number.

Example 5 Genetic Changes in Evolved E. blattae

[0071] 1,3-propanediol dehydrogenase (PDD) was compared between wildtype E. blattae and the evolved strain GEB031. The PDD from the evolvedstrain had a higher Km for 1,3-propanediol.

[0072] Materials and Methods

[0073] Strains—Wild type ATCC 33429, E. blattae comprising the mutantPDD as described in Example 4 and having ATCC accession number ______.

[0074] Growth—Cells were grown in a complex medium at 30C 500 ml in a2800 ml fernbach with shaking at 225 rpm for 20 hr. The medium consistsof KH2PO4, 5.4 g/L; (NH4)2SO4, 1.2 g/L; MgSO47H2O, 0.4 g/L; yeastextract, 2.0 g/L; tryptone, 2.0 g/L; and glycerol, 9.2 g/L in tap water.The pH was adjusted to 7.1 with KOH before autoclaving (Honda, et al.,1980, J. Bacteriol, 143:1458-1465).

[0075] Extract prep—Cells were harvested by centrifugation with care toavoid anaerobic conditions. Pellets were resuspended in 100 mM TricinepH 8.2 containing 50 mM KCl and 1 mM DTT. Cells were disrupted bypassage through a French pressure cell. Crude extracts were clarified bycentrifugation at 20K×g for 20 min followed by 100K×g for 1 hr to yieldthe high speed supernatant (HSS) fraction.

[0076] Assays—the assay for PDD was performed as described by Johnson,E. A. et al., 1987, J. Bacteriol. 169:2050-2054.

[0077] Partial purification of PDD—HSS was separated on a 16×100 Poros20 HQ column. The buffers were A, 50 mM HEPES, pH 7.4 containing 100 uMMnCl and B, A buffer containing 500 mM KCl. The column was loaded anddeveloped at 10 ml/min. The gradient was 10 CV wash, a linear gradientto 70% B in 10 CV, and 1 CV to 100% B. The activity was detected in thevery early fractions of the gradient. Pooled column fractions of the33429 strain were used as collected for assays after the addition ofadditional of DTT to 1 mM. The active fractions from strain GEB031 werepooled and concentrated on a PM30 membrane and used as concentratedafter the addition of additional 1 mM DTT. Strain GD (U/mg) PDD (U/mg)Ratio GD/PDD 33429 0.64 0.22 2.9 GEB031 0.79 0.08 9.9

[0078] PDD Kinetics-The results are shown below. Strain Km (mMPropanediol) Km (uM NAD) 33429 28 57

Example 6 Cloning and Sequencing the 1,3-propanediol Dehydrogenase Genes(dhaT) From E. blattae

[0079] The dhaT genes were amplified by PCR from genomic DNA from E.blattae as template DNA using synthetic primers (primer 1 and primer 2)based on the K. pneumoniae dhaTsequence and incorporating an Xbal siteat the 5′ end and a BamHl site at the 3′ end. The product was subclonedinto pCR-Blunt II-TOPO (Invitrogen). The cloning dhaT were thensequenced was standard techniques. The results of the DNA sequencing aregiven in SEQ ID NO:1 and SEQ ID NO:2. Primer 15′ TCTGATACGGGATCCTCAGAATGCCTGGCGGAAAAT3′ Primer 25′ GCGCCGTCTAGAATTATGAGCTATCGTATGTTTGATTATCTG3′

[0080] As will be readily understood by the skilled artisan, nucleicacid sequence generated via PCR methods may comprise inadvertent errors.The present invention also encompasses nucleic acid encoding PDDobtainable from E. blattae having ATCC accession number ______.

Example 7 Comparison of Wild-type E. blattae (ATCC Accession Number33429) and the Evolved Strain GEB031-4 (ATCC Accession Number ______).

[0081] This example shows that E. blattae subjected to the methods ofthe present invention and having ATCC accession number ______ cancompletely consume 800 mM glycerol during anaerobic fermentation anddoes not accumulate 3-hydroxy-propionaldehyde (3HPA) and does not loseviability. In contrast, the wild-type E. blattae accumulates 50 mM 3 HPAand becomes non viable after consuming only 350 mM glycerol.

[0082] The wild-type E. blattae and the evolved E. blattae weresubjected to fermentation in the following medium: 75 g glycerol, 5 gK₂HPO₄.3H₂O, 3 g KH₂PO₄, 2 g (NH₄)₂SO₄, 0.4 g MgSO₄.7H₂O, 0.2 gCaCl₂.2H₂O, 4 mg CoCl₂.2H₂O, 2 g yeast extract, and 1 g peptone perliter water. The pH was maintained with 20% NaOH. Both fermentationswere run at 30° C. with a N₂ sparge and were inoculated with astationary grown overnight preculture. The wild-type E. blattaeaccumulated 3HPA and stopped growing and metabolizing glycerol. Theaccumulation of 3HPA was high and reached 50 mM. The cell density didnot change with the culture age, but viability of the cells did. Platecounts demonstrated that accumulation of 3HPA was toxic. In contrast,the evolved strain did not accumulate more than about 6 mM 3HPA and didnot lose viability with culture age. After the culture had consumed allof the glycerol more was added and the culture continued convertingglycerol to 1,3-propanediol. See FIGS. 5 and 6. All references citedherein, including patents, patent applications, sequences andpublications are hereby incorporated in their entirety by reference.

1 15 1 741 DNA E. coli 1 atgaccgcta tgagcactgc aattacacgc cagatcgttctcgataccga aaccaccggt 60 atgaaccaga ttggtgcgca ctatgaaggc cacaagatcattgagattgg tgccgttgaa 120 gtggtgaacc gtcgcctgac gggcaataac ttccatgtttatctcaaacc cgatcggctg 180 gtggatccgg aagcctttgg cgtacatggt attgccgatgaatttttgct cgataagccc 240 acgtttgccg aagtagccga tgagttcatg gactatattcgcggcgcgga gttggtgatc 300 cataacgcag cgttcgatat cggctttatg gactacgagttttcgttgct taagcgcgat 360 attccgaaga ccaatacttt ctgtaaggtc accgatagccttgcggtggc gaggaaaatg 420 tttcccggta agcgcaacag cctcgatgcg ttatgtgctcgctacgaaat agataacagt 480 aaacgaacgc tgcacggggc attactcgat gcccagatccttgcggaagt ttatctggcg 540 atgaccggtg gtcaaacgtc gatggctttt gcgatggaaggagagacaca acagcaacaa 600 ggtgaagcaa caattcagcg cattgtacgt caggcaagtaagttacgcgt tgtttttgcg 660 acagatgaag agattgcagc tcatgaagcc cgtctcgatctggtgcagaa gaaaggcgga 720 agttgcctct ggcgagcata a 741 2 246 PRT E. coli2 Met Thr Ala Met Ser Thr Ala Ile Thr Arg Gln Ile Val Leu Asp Thr 1 5 1015 Glu Thr Thr Gly Met Asn Gln Ile Gly Ala His Tyr Glu Gly His Lys 20 2530 Ile Ile Glu Ile Gly Ala Val Glu Val Val Asn Arg Arg Leu Thr Gly 35 4045 Asn Asn Phe His Val Tyr Leu Lys Pro Asp Arg Leu Val Asp Pro Glu 50 5560 Ala Phe Gly Val His Gly Ile Ala Asp Glu Phe Leu Leu Asp Lys Pro 65 7075 80 Thr Phe Ala Glu Val Ala Asp Glu Phe Met Asp Tyr Ile Arg Gly Ala 8590 95 Glu Leu Val Ile His Asn Ala Ala Phe Asp Ile Gly Phe Met Asp Tyr100 105 110 Glu Phe Ser Leu Leu Lys Arg Asp Ile Pro Lys Thr Asn Thr PheCys 115 120 125 Lys Val Thr Asp Ser Leu Ala Val Ala Arg Lys Met Phe ProGly Lys 130 135 140 Arg Asn Ser Leu Asp Ala Leu Cys Ala Arg Tyr Glu IleAsp Asn Ser 145 150 155 160 Lys Arg Thr Leu His Gly Ala Leu Leu Asp AlaGln Ile Leu Ala Glu 165 170 175 Val Tyr Leu Ala Met Thr Gly Gly Gln ThrSer Met Ala Phe Ala Met 180 185 190 Glu Gly Glu Thr Gln Gln Gln Gln GlyGlu Ala Thr Ile Gln Arg Ile 195 200 205 Val Arg Gln Ala Ser Lys Leu ArgVal Val Phe Ala Thr Asp Glu Glu 210 215 220 Ile Ala Ala His Glu Ala ArgLeu Asp Leu Val Gln Lys Lys Gly Gly 225 230 235 240 Ser Cys Leu Trp ArgAla 245 3 1164 DNA E. blatte 3 atgagctatc gtatgtttga ttatctggttccaaatgtga acttctttgg cccgggcgcc 60 gtttctgttg ttggccagcg ctgccagctgctggggggta aaaaagccct gctggtgacc 120 gataagggcc tgcgcgccat taaagacggtgctgtcgatc agaccgtgaa gcacctgaaa 180 gccgccggta ttgaggtggt cattttcgacggggtcgagc cgaacccgaa agacaccaac 240 gtgctcgacg gcctggccat gttccgtaaagagcagtgcg acatgataat caccgtcggc 300 ggcggcagcc cgcacgactg cggtaaaggcattggtattg cggccaccca cccgggtgat 360 ctgtacagct atgccggtat cgaaacactcaccaacccgc tgccgcccat tattgcggtc 420 aacaccaccg ccgggaccgc cagcgaagtcacccgccact gcgtgctgac taacaccaaa 480 accaaagtaa aatttgtgat tgtcagctggcgcaacctgc cttccgtctc cattaacgat 540 ccgctgctga tgatcggcaa gcccgccgggctgaccgccg ccaccggtat ggatgccctg 600 acccacgcgg tagaggccta tatctccaaagacgccaacc cggttaccga tgcctctgct 660 attcaggcca tcaaactgat tgccaccaacttgcgccagg ccgtcgccct ggggaccaac 720 ctcaaagccc gtgaaaacat ggcctgcgcctctctgctgg ccgggatggc ctttaacaac 780 gccaacctgg gctatgttca cgccatggctcaccagctgg gcggcctgta cgacatggcc 840 cacggggtgg cgaacgcggt cctgctgccccatgtctgcc gctataacct gattgccaac 900 ccggaaaaat ttgccgatat cgccacctttatgggggaaa acaccaccgg tctttccacc 960 atggacgcag cggagctggc catcagcgccattgcccgtc tgtctaaaga tgtcgggatc 1020 ccgcagcacc tgcgtgaact gggggtaaaagaggccgact tcccgtacat ggcagaaatg 1080 gccctgaaag acggcaacgc cttctctaacccgcgcaaag ggaacgaaaa agagattgcc 1140 gacattttcc gccaggcatt ctga 1164 4387 PRT E. blatte 4 Met Ser Tyr Arg Met Phe Asp Tyr Leu Val Pro Asn ValAsn Phe Phe 1 5 10 15 Gly Pro Gly Ala Val Ser Val Val Gly Gln Arg CysGln Leu Leu Gly 20 25 30 Gly Lys Lys Ala Leu Leu Val Thr Asp Lys Gly LeuArg Ala Ile Lys 35 40 45 Asp Gly Ala Val Asp Gln Thr Val Lys His Leu LysAla Ala Gly Ile 50 55 60 Glu Val Val Ile Phe Asp Gly Val Glu Pro Asn ProLys Asp Thr Asn 65 70 75 80 Val Leu Asp Gly Leu Ala Met Phe Arg Lys GluGln Cys Asp Met Ile 85 90 95 Ile Thr Val Gly Gly Gly Ser Pro His Asp CysGly Lys Gly Ile Gly 100 105 110 Ile Ala Ala Thr His Pro Gly Asp Leu TyrSer Tyr Ala Gly Ile Glu 115 120 125 Thr Leu Thr Asn Pro Leu Pro Pro IleIle Ala Val Asn Thr Thr Ala 130 135 140 Gly Thr Ala Ser Glu Val Thr ArgHis Cys Val Leu Thr Asn Thr Lys 145 150 155 160 Thr Lys Val Lys Phe ValIle Val Ser Trp Arg Asn Leu Pro Ser Val 165 170 175 Ser Ile Asn Asp ProLeu Leu Met Ile Gly Lys Pro Ala Gly Leu Thr 180 185 190 Ala Ala Thr GlyMet Asp Ala Leu Thr His Ala Val Glu Ala Tyr Ile 195 200 205 Ser Lys AspAla Asn Pro Val Thr Asp Ala Ser Ala Ile Gln Ala Ile 210 215 220 Lys LeuIle Ala Thr Asn Leu Arg Gln Ala Val Ala Leu Gly Thr Asn 225 230 235 240Leu Lys Ala Arg Glu Asn Met Ala Cys Ala Ser Leu Leu Ala Gly Met 245 250255 Ala Phe Asn Asn Ala Asn Leu Gly Tyr Val His Ala Met Ala His Gln 260265 270 Leu Gly Gly Leu Tyr Asp Met Ala His Gly Val Ala Asn Ala Val Leu275 280 285 Leu Pro His Val Cys Arg Tyr Asn Leu Ile Ala Asn Pro Glu LysPhe 290 295 300 Ala Asp Ile Ala Thr Phe Met Gly Glu Asn Thr Thr Gly LeuSer Thr 305 310 315 320 Met Asp Ala Ala Glu Leu Ala Ile Ser Ala Ile AlaArg Leu Ser Lys 325 330 335 Asp Val Gly Ile Pro Gln His Leu Arg Glu LeuGly Val Lys Glu Ala 340 345 350 Asp Phe Pro Tyr Met Ala Glu Met Ala LeuLys Asp Gly Asn Ala Phe 355 360 365 Ser Asn Pro Arg Lys Gly Asn Glu LysGlu Ile Ala Asp Ile Phe Arg 370 375 380 Gln Ala Phe 385 5 12 DNAArtificial Sequence wild type mutD gene 5 atgaccgcta tg 12 6 11 DNAArtificial Sequence pOS100 mutD mutated gene 6 ttgacgcttt g 11 7 12 DNAArtificial Sequence pOS101 mutD mutated gene 7 gtgaccgctg tg 12 8 11 DNAArtificial Sequence pOS102 mutD mutated gene 8 gtgccgctgt g 11 9 12 DNAArtificial Sequence pOS104 mutD mutated gene 9 ttgaccgctt tg 12 10 55DNA Artificial Sequence pOS105 mutD mutated gene 10 gtgaccgctgtgagcacttg caattacacg ccagatcgtt ctcgataccg aaatc 55 11 11 DNAArtificial Sequence pOS106 mutD mutated gene 11 gtgaccgctt g 11 12 28DNA Artificial Sequence primer 12 cgcctccagc gcgacaatag cggccatc 28 1327 DNA Artificial Sequence primer 13 ccgactgaac taccgctccg cgttgtg 27 1436 DNA Artificial Sequence primer 14 tctgatacgg gatcctcaga atgcctggcggaaaat 36 15 42 DNA Artificial Sequence primer 15 gcgccgtcta gaattatgagctatcgtatg tttgattatc tg 42

We claim:
 1. A method for preparing an evolved microorganism comprisingthe steps of: a. culturing a microorganism comprising at least oneheterologous mutator gene for at least 20 doublings under conditionssuitable for selection of an evolved microorganism, wherein saidheterologous mutator gene generates a mutation rate of at least5-100,000 fold relative to wild type, and b. restoring said evolvedmicroorganism to a wild type mutation rate.
 2. The method of claim 1wherein said microorganism further comprises at least one introducednucleic acid encoding a heterologous protein.
 3. The method of claim 2wherein said heterologous protein(s) includes hormones, enzymes andgrowth factors.
 4. The method of claim 3 wherein said heterologousprotein is an enzyme.
 5. The method of claim 4 wherein said enzymeincludes hydrolases, such as protease, esterase, lipase, phenol oxidase,permease, amylase, pullulanase, cellulase, glucose isomerase, laccaseand protein disulfide isomerase.
 6. The method of claim 1 wherein saidmicroorganism further comprises introduced nucleic acid encoding atleast one enzyme necessary for an enzymatic pathway.
 7. The method ofclaim 6 wherein said enzyme is a reductase or a dehydrogenase and saidenzymatic pathway is for the production of ascorbic acid or ascorbicacid intermediates.
 8. The method of claim 6 wherein said enzyme isglycerol dehydratase or 1,3-propanediol dehydrogenase and said enzymaticpathway is for the production of 1,3 propanediol, 1,3 propanediolprecursors or 1,3 propanediol derivatives.
 9. The method of claim 6wherein said enzyme is glycerol-3-phosphate dehydrogenase orglycerol-3-phosphate phosphatase and said pathway is for the productionof glycerol and glycerol derivatives.
 10. The method of claim 6 whereinsaid enzymatic pathway is for the production of amino acids or dyes. 11.The method of claim 1 wherein said microorganism is cultured for betweenabout 20 to about 100 doublings.
 12. The method of claim 1 wherein saidmicroorganism is cultured for between about 100 to about 500 doublings.13. The method of claim 1 wherein said microorganism is cultured forbetween about 500 to about 2000 doublings.
 14. The method of claim 1wherein said microorganism is cultured for greater than 2000 doublings.15. The method of claim 1 wherein said evolved microorganism comprisesfrom about 3 to about 1000 selected mutations.
 16. The method of claim 1wherein said evolved microorganism further comprises from about 20 toabout 100,000 neutral mutations
 17. The method of claim 1 wherein saidevolved microorganism comprises about 3 to about 1000 selected mutationsin about 3 to about 500 genes.
 18. The method of claim 17 wherein saidmutations are non-specific.
 19. The method of claim 17 wherein said mutat ions are specific.
 20. The method of claim 1 wherein said mutatorgene generates a mutation rate of at least about 5 fold to about 10,000fold relative to wild type.
 21. The method of claim 1 wherein saidmutator gene generates a mutation rate of at least about 5 fold to about1000 fold.
 22. The method of claim 1 wherein said mutator gene generatesa mutation rate of about 5 fold to about 1000 fold over wild type. 23.The method of claim 1 wherein said microorganism comprises a plasmidcomprising the heterologous mutator gene and said step of restoring saidevolved microorganism to a wild type mutation rate comprises curing theevolved microorganism of said plasmid.
 24. The method of claim 23wherein said plasmid comprises a temperature sensitive origin ofreplication.
 25. The method of claim 1 wherein said microorganismcomprises at least one copy of the mutator gene in the chromosome andsaid step of restoring said evolved microorganism to wild type mutationrate comprise excision of said mutator gene.
 26. The method of claim 1wherein said mutator gene comprises mutD, mutT, mutY, mutM, mutH, mutL,mutS or mutU mutations or homologues thereof.
 27. The method of claim 26wherein said mutator gene comprises mutD having mutations shown in TableI.
 28. The method of claim 1 wherein said conditions suitable forselection comprise culturing said microorganism in the presence of atleast one organic solvent.
 29. The method of claim 28 wherein saidorganic solvent includes alcohols, diols, hydrocarbon, mineral oil,mineral oil derived products, halogenated compounds and aromaticcompounds.
 30. The method of claim 1 wherein said conditions suitablefor selection comprise culturing said microorganism in the presence ofelevated temperature.
 31. The method of claim 30 wherein said elevatedtemperature is about 42° C. to about 48° C.
 32. The method of claim 1wherein said conditions suitable for selection comprise culturing saidmicroorganism in the presence of high salt.
 33. The method of claim 1wherein said microorganism includes Gram-positive or a Gram-negativemicroorganism, fungus, yeast or eucaryotic.
 34. The method of claim 33wherein said microorganism is an Enterobacteriaceae.
 35. The method ofclaim 34 wherein said microorganism is an Eschericia.
 36. The method ofclaim 35 wherein said microorganism is E. coli.
 37. The method of claim35 wherein said microorganism is E. blatte.
 38. The method of claim 1wherein said evolved microorganism is E. coli having ATCC accessionnumber ______.
 39. The method of claim 1 wherein said evolvedmicroorganism is E. blattae having ATCC accession number
 40. Anexpression vector comprising a mutator gene.
 41. The expression vectorof claim 40 wherein said mutator gene is a mutated MutD.
 42. Theexpression vector of claim 40 wherein said mutated MutD has themutations as shown in Table I.
 43. A host cell comprising the expressionvector of claim
 40. 44. The host cell of claim 43 that is aGram-positive or Gram-negative microorganism.
 45. The host cell of claim44 that is an Enterobacteriaceae.
 46. The isolated E. blattaemicroorganism deposited with the ATCC and having accession number. 47.The isolated E. coli microorganism deposited with the ATCC and havingaccession number.
 48. A method for preparing an evolved microorganismcomprising the steps of: a. mutating a DNA repair gene in amicroorganism to obtain a mutated strain, b. culturing the mutatedstrain for at least 20 doublings under conditions suitable for selectionof an evolved strain, wherein said mutated strain generates a mutationrate of at least 5-100,000 fold relative to the wild-type microorganism,and c. restoring the naturally occurring DNA repair gene in said evolvedmicroorganism.